In heart cells, a major route for Ca2+ to move across the plasma membrane is through the highly specialized protein called the Na+-Ca2+ exchanger (NCX). This transporter moves Ca2+ against its gradient using the electrochemical gradient of Na+ as driving force. By maintaining Ca2+ homeostasis, NCX regulates cardiac function and alterations in its activity or expression are arrhythmogenic. Accordingly, NCX inhibitors have been suggested as therapy for arrhythmia, providing strong motivation for learning more about the structure and regulation of NCX. This is needed as, in spite of the recently resolved crystal structure of an archaebacterial homolog, salient features of this important transporter are still unknown. As a consequence, the regions of the protein involved in ion transport are not well defined and the molecular mechanisms of ion translocation are unknown. Also unclear is the physiological role of Ca2+ regulation since it is difficult to distinguish the role of Ca2+ as an activator and as transport substrate in intact cells. Another feature of NCX that has been largely overlooked is the functional role of its dimeric state and whether this property alters excitation contraction coupling is still uncharted. The objective of this proposal is to improve our understanding of NCX structure and regulation. To accomplish this we have combined cutting edges tools such as mutagenesis, electrophysiology and optical techniques, including a newly developed approach to measure conformational changes of plasma membrane proteins by FRET. Three specific aims are proposed. Aim1 will map the residues of the protein involved with ion translocation by utilizing cysteine scanning mutagenesis and electrophysiology. We will also identify and characterize the local rearrangement of NCX transmembrane segments (TMS) occurring during ion translocation using Voltage Clamp Fluorometry. This is a sophisticated and very powerful technique which permits to resolve in real time membrane protein motion (detected as changes in fluorescence) and the associated ionic flux, under voltage clamp. Aim2 will focus on elucidating if in vivo cytoplasmic Ca2+ modulates NCX activity preferentially via its allosteric regulation or transport site. These studies will resolve a major controversy in the fiel. Finally, Aim3 will define for the first time if NCX dimerizes in heart cells and determine whether NCX oligomerization is necessary for transport or regulation. This research proposal will better define the role of NCX in physiological and pathophysiological conditions and help in the rational design of drugs targeting NCX, which have promising therapeutic effects. Their development has been markedly limited by the lack of structural and mechanistic information on NCX. The studies outlined herein will help in this endeavor and greatly advance our understanding of NCX.